Diagnostic performance of respirators for collection and detection of SARS-CoV-2

Respirators, called as face mask, have been used to protect the wearer from the outside harmful air environment and prevent any virus from being released to neighbors from potentially infected exhaled breath. The antiviral effectiveness of respirators has not only been researched scientifically, but has also become a global issue due to society's obligation to wear respirators. In this paper, we report the results of a study on the collection and detection of viruses contained in exhaled breath using respirators. The inner electrostatic filter was carefully selected for virus collection because it does not come in direct contact with either human skin or the external environment. In the study of a healthy control group, it was confirmed that a large amount of DNA and biomolecules such as exosomes were collected from the respirator exposed to exhalation, and the amount of collection increased in proportion to the wearing time. We conducted experiments using a total of 72 paired samples with nasopharyngeal swabs and respirator samples. Out of these samples, fifty tested positive for SARS-CoV-2 and twenty-two tested negative. The PCR results of the NPS and respirator samples showed a high level of agreement, with a positive percent agreement of ≥ 90% and a negative percent agreement of ≥ 99%. Furthermore, there was a notable level of concordance between RCA-flow tests and PCR when examining the respirator samples. These results suggest that this is a non-invasive, quick and easy method of collecting samples from subjects using a respirator, which can significantly reduce the hassle of waiting at airports or public places and concerns about cross-contamination. Furthermore, we expect miniaturized technologies to integrate PCR detection into respirators in the near future.


Table S1
List of SARS-CoV-2 template, pathogen, linker primer, and primer nucleic acid sequences used in this study Table S2 Diagnostic criteria by flow in RCA-flow system.

Immobilization of Linker Primer on Nylon Mesh Surface
After the washing step, 200 μL of 100-mM NHS buffer and 200 μL of 100-mM WSC buffer (both from the coupling kit) were mixed with nylon mesh and incubated at room temperature (~23 °C) for 30 min. The nylon mesh was then washed twice with 500 μL of 1×PBS buffer. The washed nylon mesh was then resuspended in 380 μL of reaction buffer and 20 μL of 1-mM NH2 primer and incubated for 2 h. The nylon mesh was then washed twice with 500 μL of 1×PBS buffer, resuspended in 500 μL of blocking buffer and incubated for 1 h. The nylon mesh was washed twice and resuspended in 400 μL of 1×PBS buffer.

Extraction of SARS-CoV-2 virus nucleic acid from face mask
The RNA extraction process from the mask involved the following steps. Initially, the facemask was carefully opened, and the filter membrane was extracted. Subsequently, the membrane was cut into a size of 50 mm x 50 mm and placed inside a 10 mL syringe for sample extraction. 5 mL of Trizol was then dispensed into the syringe, ensuring that it covered the membrane of the mask.
The syringe with the Trizol-covered membrane was incubated at room temperature for 10 minutes.
After incubation, the entire solution was withdrawn from the syringe and divided into 1 mL tubes each. Then, 200 μl of chloroform was dispensed into the 1 ml solution. It was then shaken for 15 seconds and incubated for 5 minutes at room temperature. After centrifugation at 12,000 g, 4° C., for 10 minutes, only the transparent supernatant was separated. After mixing 500 µl of isopropanol with the supernatant, we incubated it for 10 min at room temperature after stirring. It was centrifuged again at 12,000 g, 4 °C, 10 min, and then the supernatant was removed. After adding 1 ml of 75% ethanol to the RNA from which the supernatant was removed, we it centrifuged at 7500 g, 4 °C, 5 min, and then removed the supernatant (ethanol). It was dried (to remove ethanol completely) for 5--10 min with the lid open, and washing was performed twice depending on the sample. Finally, after dispensing 15 µl of RNase-free water (DW) and pipetting, we incubate it at 65 °C for 10 min in a heating block, and then incubate it on ice for 2 min to extract RNA.

Increase in the amount of recovery of viral nucleic acids from the filter layer
To enhance the recovery of viral nucleic acids from the filter layers, we utilized a centrifuge with a syringe. Initially, the mask membrane was cut to the appropriate size (as depicted in Fig. S1(a)-S1(d)). Subsequently, the mask membrane was incubated in 5 ml of Trizol at room temperature for 10 minutes and then placed inside the body of a 10 ml syringe. The syringe body, containing the mask membrane, was inserted into a 50 ml tube. Using a centrifuge set at 7000 g for 15 minutes, the entire solution was extracted from the mask.
When employing 5 ml of lysis buffer within the mask, approximately 4-4.4 ml (80%-86%) of the solution could be recovered using a syringe. However, by utilizing the centrifuge method, over 4.8 ml (95%) of the solution could be successfully recovered. The extraction protocol plays a crucial role in increasing the concentration of viral nucleic acids and addressing the concentration-related issues. Higher recovery rates of the mask lysis solution, exceeding 95%, can potentially lead to an increase in concentration. As demonstrated in Fig. S2(e), we evaluated the recovery volume of the lysis solution using both the syringe and centrifuge methods. The centrifugation technique yielded a relatively higher recovery volume. Furthermore, as shown in Fig. S2(f), we were able to quantify the nucleic acids targeting viral genes through RT-qPCR and compare their Ct values using the centrifuged samples, which exhibited relatively higher concentrations.

Validation of DNA hydrogel formation by SARS-CoV-2 templates
To prepare the RCA mixture, we obtained 1 μL of 100 μM clinical mask sample (containing RNA), 1 μL of T4 ligase (400 U/μL), 1 μL of pyrophosphatase (100 U/mL), 4 μL of phi29 DNA polymerase (10 U/μL), and 40 μL of a 10x phi29 reaction buffer. The 10x phi29 reaction buffer consisted of 4 μL of polymerase buffer, 1 μL of 100 mM DTT, 4 μL of 25 mM dNTP, 1 μL of 50 mM ATP, 1 μL of 100 μM additional primer, 1 μL of BSA (10 mg/mL), and 21 μL of distilled water. To confirm the formation of DNA hydrogels, we added various types of SARS-CoV-2 templates to the RCA mixture and incubated it at 30 °C for 60 minutes. The dumbbell-shaped templates have a tendency to promote entanglement and aggregation with neighboring DNAs, resulting in the formation of DNA hydrogels.After incubation, we observed the formation of significant DNA hydrogels in the tubes containing the N, E, and RdRp gene templates of SARS-CoV-2. In contrast, no DNA hydrogel formation was observed in the tube without the SARS-CoV-2 template. The captured image (Fig. S3) clearly demonstrates the strong formation of DNA hydrogels depending on the presence of the target template.

Comparison of quantitative values for each SARS-CoV-2 target gene according to the date of mask collection
Analysis was conducted using a total of N=9 masks collected over a span of three days. Quantification was performed through RT-PCR, targeting the SARS-CoV-2 genes (E, RdRp, and N genes). Fig. S4 (a) to (c) presents the results, indicating that the overall Ct value for each target gene exhibited a trend of shifting towards higher values over time. This observation suggests that the viral load tended to decrease as the days of collection progressed.

Comparison of quantitative values for SARS-CoV-2 target gene according to each sample
Analysis was conducted on each sample type (nasal swab, saliva, mask) with N=3 samples for each type. Virus quantification was carried out using RT-PCR, employing the target genes of SARS-CoV-2 (E, RdRp, N genes). Fig. S5 (a) to (c) illustrates the results obtained, indicating that there was no significant variation in the Ct value across the different target genes based on the sample type. This suggests that the efficacy of nucleic acid collection from the mask is comparable to that of conventional sample collection methods. Therefore, our findings demonstrate that the performance of mask-based sample collection is not significantly inferior to traditional approaches.  Table S1. List of SARS-CoV-2 template, pathogen, linker primer, and primer nucleic acid sequences used in this study.